Embryonic cells select different pathways of differentiation as a result of their locations within the developing embryo. For Drosophila, most of the genetic loci that specify this positional information have been identified. Some of these genes contain a similar 180 bp protein coding region designated the homeo box. Because homeo box protein domains appear to contain sequence-specific DNA binding activities, it is possible that homeo box genes control embryonic development by the trans regulation of "batteries" of target genes. There are at least 20 homeo box genes in Drosophila. To date, 14 of these gene have been cloned and characterized. In most cases the cloned genes derive from genetic loci that are involved in the elaboration of positional identity along the anterior-posterior (A-P) body axis of developing embryos. However, at least two of the genes appear to be required for the differentiation of the dorsal-ventral (D-V) pattern. Each homeo box gene shows a unique spatial and temporal pattern of expression during development. A central problem in the control of positional information is how these different genes come to be expressed in specific regions of the developing embryo. There is genetic evidence that hierarchical cross-regulatory interactions among homeo box genes play an important role in the initiation or maintenance of their selective patterns of expression. The overall goal of the proposed research is to elucidate mechanisms responsible for the transcriptional control of homeo box gene expression. Towards this end we will analyze regulatory interactions among a set of Drosophila homeo box genes that are active during early embryonic development. This study will involve: i) Preparation of high-affinity antibodies that recognize proteins encoded by homeo box genes; ii) Isolation and characterization of "native" homeo box proteins; iii) The identification of cis regulatory sequences required for homeo box gene expression by P-mediated gene transfer; iv) DNA binding and footprint analyses using cloned homeo box genes and purified "native" proteins; v) In vitro transcription assays of homeo box gene expression using Drosophila and HeLa extracts; and vi) Co-transfection of cultured cell lines with combinations of cloned homeo box genes.